Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

• the present invention relates to compounds useful for the modulation of the activity of Stimulator of Interferon Genes (STING) and particularly, although not exclusively, to proteolysis targeting chimeric (PROTAC ® ) compounds (also referred to herein as degraders/degrader compounds) which induce degradation of STING by binding to both STING and an E3 ubiquitin ligase, and uses of such compounds.
• STING Stimulator of Interferon Genes
• PROTAC ® proteolysis targeting chimeric compounds
• the degrader binds to both the target protein and E3 ubiquitin ligase simultaneously to form a ternary complex.
• the E3 ligase then recruits an E2 conjugating enzyme to the ternary complex, which ubiquitinates the target protein. This has the effect of labelling the target protein for degradation by the cell's proteasome machinery.
• the degrader can then dissociate from the target protein and initiate another cycle of this process in a catalytic manner. Meanwhile, the ubiquitinated target proteins are recognized and degraded by the cell's proteasome machinery.
• Cereblon is a substrate specificity receptor that associates with the Cullin-4-RING E3 ubiquitin ligase (CRL4) complex.
• Known ligands for CRBN include thalidomide and other immunomodulatory imide drugs (IMiDs).
• IiDs immunomodulatory imide drugs
• CRBN has been widely used as an E3 ligase in degrader compounds targeting a range of proteins.
• the Inhibitor of Apoptosis Proteins (lAPs) family includes antiapoptotic proteins, which are commonly overexpressed in some cancer cells and promote their survival, as well as the survival of neuronal cells.
• certain lAPs possess E3 ubiquitin ligase activity, such as cellular lAPs 1 and 2 (clAP-1/2) and ligands for these molecules have been incorporated into the design of degrader compounds.
• STING Stimulator of Interferon Genes
• IFN type I interferon
• Cytosolic STING is activated upon binding cyclic dinucleotides (CDNs), which can be derived from bacterial sources or 2',3'-cyclic GMP-AMP (2',3'-cGAMP) generated by cyclic GMP-AMP synthase (cGAS), an enzyme that is activated by binding to double-stranded DNA in the cytoplasm.
• CDNs cyclic dinucleotides
• cGAS cyclic GMP-AMP synthase
• STING can also be activated in a non-canonical CDN-independent manner, for example through gain-of-function mutations (Decout et al., 2021).
• STING leads to up-regulation of type I interferon production.
• Activation of type I interferon production is an approach which has been explored in the treatment of some cancers, and numerous STING agonists have been designed for this purpose.
• STING agonists include benzimidazoles, for example as described in WO2017/175147 , WO2019/069270 , WO2020/132582 , WO2020/132566 , and WO2020/132549 the content of each of which is hereby incorporated by reference.
• WO2013/106643 describes compounds and methods for the enhanced degradation of targeted proteins and other polypeptides by an E3 ubiquitin ligase.
• WO2016/197032 describes imide-based compounds, including bifunctional compounds comprising the same, which find utility as modulators of targeted ubiquitination, especially inhibitors of a variety of polypeptides and other proteins which are degraded and/or otherwise inhibited by bifunctional compounds.
• WO2016/169989 describes PROTAC ® /degrader compounds incorporating a selective E3 Ligase IAP binding moiety (IAP binding moiety) as the degradation component, functioning to recruit target proteins to the E3 ubiquitin ligase IAP for degradation.
• IAP binding moiety selective E3 Ligase IAP binding moiety
• WO2018/066545 describes drugs that can decompose a target intracellular protein specifically and is effective for the prevention or treatment of diseases associated with the target protein.
• a SNIPER compound produced by linking a specific IAP ligand, through a linker, to a ligand capable of specifically binding to a target intracellular protein.
• the autoimmune disease systemic lupus erythematosus has also been linked with cGAS pathway activity and type I interferon induction via STING, providing a mechanism for how cGAS-STING may amplify or accelerate disease symptoms.
• Sjögren's syndrome is a further autoimmune disorder characterized by an increased type 1 interferon gene signature (Decout et al., 2021).
• Inflammation is also a prominent hallmark of several neurodegenerative diseases, and elevated levels of type I interferons and contributions of STING have also been observed in models and disease samples of Huntington disease (Decout et al., 2021).
• STING antagonists rely on high systemic drug exposures to maintain sufficient STING inhibition, which increases the risk of unwanted off-target effects.
• existing STING antagonists are less effective in the treatment of diseases driven by constitutive (agonist-free) activation of STING.
• the present invention provides a use of the compound of the first aspect, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or condition in a subject in need thereof.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising the compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
• the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
• substituted refers to a parent group which bears one or more substituents.
• substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group.
• substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
• halogen or halo is selected from chloro (Cl), fluoro (F), bromo (Br) and iodo (I), such as fluoro.
• C 2-8 alkenyl The term "C 2-8 alkenyl" as used herein, pertains to a non-aromatic hydrocarbon group having one or more carbon-carbon double bonds.
• C 4-12 heterocyclyl refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 4 to 12 ring atoms, of which from 1 to 5 are ring heteroatoms. In certain embodiments, each ring has from 5 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
• the ring may be saturated or unsaturated and may be bridged or unbridged.
• the ring may be a fused ring or a single ring. For the avoidance of doubt, substituents on the heterocyclyl ring may be linked via either a carbon atom or a heteroatom.
• the prefixes e.g. C 5-10 C 5-7 , C 5-6 , etc.
• the term "C 5-6 heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
• monocyclic heterocyclyl groups include, but are not limited to, those derived from:
• bicyclic heterocyclyl groups include, but are not limited to those derived from:
• carboaryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C 9 ), indene (C 9 ), isoindene (C 9 ) and tetraline (1,2,3,4-tetrahydronaphthalene) (C 10 ).
• indane e.g. 2,3-dihydro-1H-indene
• indene C 9
• isoindene C 9
• tetraline (1,2,3,4-tetrahydronaphthalene
• the selected substituents may comprise the same substituents or different substituents from within the given group.
• the degrader/PROTAC ® linker connects the two functional motifs of a degrader/PROTAC ® , a target protein binder and an E3 ligase recruiter.
• An optimal linker will allow for maximal interaction between the target protein and the E3 ligase resulting in efficient ubiquitination of the target protein and its ultimate degradation.
• the linker length can vary from 1-45 atoms in length.
• the length and chemical composition of the linker affects the structural rigidity, hydrophobicity and solubility of a degrader/PROTAC ® .
• the structure of the linker is described in more detail below.
• a suitable pharmaceutically acceptable salt of a compound of Formula (I-b) is, for example, an acid-addition salt.
• An acid addition salt of a compound of Formula (I-b) may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person.
• An acid addition salt may for example be formed using an inorganic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid.
• An acid addition salt may also be formed using an organic acid selected from the group consisting of trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
• solvated forms may be a hydrated form, such as a hemihydrate, a monohydrate, a dihydrate, a trihydrate or an alternative quantity thereof.
• the compounds of Formula (I-b) encompass all such solvated and unsolvated forms of compounds of Formula (I-b), particularly to the extent that such forms possess STING antagonist activity, as for example measured using the tests described herein.
• a particular enantiomer of a compound described herein may be more active than other enantiomers of the same compound.
• a compound of Formula (I-b), or a pharmaceutically acceptable salt thereof which is a single enantiomer being in an enantiomeric excess (%ee) of ⁇ 95, ⁇ 98% or ⁇ 99%.
• the single enantiomer is present in an enantiomeric excess (%ee) of ⁇ 99%.
• a pharmaceutical composition which comprises a compound of Formula (I-b), which is a single enantiomer being in an enantiomeric excess (%ee) of ⁇ 95, ⁇ 98% or ⁇ 99% or a pharmaceutically acceptable salt thereof, in association with one or more pharmaceutically acceptable excipients.
• the single enantiomer is present in an enantiomeric excess (%ee) of ⁇ 99%.
• Atoms of the compounds and salts described in this specification may exist as their isotopes.
• the compound of Formula (I-1) encompasses all compounds of Formula (I-1) where an atom is replaced by one or more of its isotopes (for example a compound of Formula (I-1) where one or more carbon atom is an 11 C or 13 C carbon isotope, or where one or more hydrogen atoms is a 2 H or 3 H isotope).
• Tautomers are structural isomers that exist in equilibrium resulting from the migration of a hydrogen atom.
• the compound of Formula (I-1) includes all tautomers of compounds of Formula (I-1) particularly to the extent that such tautomers possess STING antagonist activity.
• Compounds and salts described in this specification may be crystalline and may exhibit one or more crystalline forms.
• the compound of Formula (I-1) encompasses any crystalline or amorphous form of a compound of Formula (I-1), or mixture of such forms, which possesses STING antagonist activity.
• compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous or intramuscular dosing), or as a suppository for rectal dosing.
• the compositions may be obtained by conventional procedures well known in the art.
• Compositions intended for oral use may contain additional components, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
• Suitable daily doses of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof, in therapeutic treatment of humans are about 0.0001-100 mg/kg body weight.
• a pharmaceutical composition for use in the treatment of a disease associated with overactivation of STING comprising a compound of Formula (I-1), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
• a pharmaceutical composition for use in the treatment of a disease comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
• a pharmaceutical composition for use in the treatment of a disease associated with overactivation of STING comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
• the E3 ubiquitin ligase ligand may optionally not be an Inhibitor of Apoptosis (IAP) E3 ubiquitin ligase ligand.
• a method of treatment comprising administering to a subject the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula (I).
• a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament comprising administering to a subject the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula (I).
• a compound of Formula (I), or a pharmaceutically acceptable salt thereof for use as a medicament.
• a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula (I) for use in treating a disorder in one embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula (I) for use in treating a disorder.
• a method of treating a disorder or condition comprising administering to a subject the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula (I).
• the disorder or condition is associated with overactivation of STING. In some embodiments, the disorder or condition is driven by constitutive STING activation. In some embodiments, the disorder or condition is driven by constitutive STING activation caused by a gain-of-function mutation.
• the E3 ubiquitin ligase ligand may optionally not be an Inhibitor of Apoptosis (IAP) E3 ubiquitin ligase ligand. In some embodiments, upon non-covalent binding of S1 to STING and E to an E3 ubiquitin ligase, STING is degraded.
• the disorder or condition is a monogenic autoinflammatory syndrome, autoimmune disease, inflammatory disease, neurological disorder, metabolic disease, cardiovascular disease, or cancer.
• the E3 ubiquitin ligase ligand may optionally not be an Inhibitor of Apoptosis (IAP) E3 ubiquitin ligase ligand.
• the disorder or condition is a monogenic autoinflammatory syndrome.
• the disorder or condition is STING-associated vasculopathy with onset in infancy (SAVI), Aicardi-Goutieres Syndrome, COPA Syndrome, Familial Chilblain Lupus, Dnase II deficiency, or TREX1 deficiency.
• the E3 ubiquitin ligase ligand may optionally not be an Inhibitor of Apoptosis (IAP) E3 ubiquitin ligase ligand.
• the disorder or condition is an autoimmune disease.
• the disorder or condition is systemic lupus erythematosus (SLE), Sjögren's syndrome, or rheumatoid arthritis.
• the disorder or condition is systemic lupus erythematosus (SLE).
• the disorder or condition is an inflammatory disease. In some embodiments, the disorder or condition is silica-induced fibrosis or sepsis.
• the disorder or condition is a neurological disorder.
• the disorder or condition is ischaemic brain injury, Parkinson disease, general neurodegeneration, Huntington disease, amyotrophic lateral sclerosis and frontotemporal dementia, age-dependent macular degeneration, or traumatic brain injury.
• the disorder or condition is a metabolic disease.
• the disease is non-alcoholic steatohepatitis, alcoholic liver disease, or acute pancreatitis.
• the disorder or condition is a cardiovascular disease. In some embodiments, the disorder or condition is myocardial infarction or chronic heart failure.
• the disorder or condition is cancer. In some embodiments, the disorder or condition is colorectal cancer, skin cancer, or metastases.
• the disorder or condition is selected from monogenic autoinflammatory syndrome, STING-associated vasculopathy with onset in infancy (SAVI), Aicardi-Goutieres Syndrome, COPA Syndrome, Familial Chilblain Lupus, DNase II deficiency, or TREX1 deficiency, an autoimmune disease, systemic lupus erythematosus (SLE), Sjögren's syndrome or a cardiovascular disease.
• SAVI STING-associated vasculopathy with onset in infancy
• COPA Syndrome Familial Chilblain Lupus
• DNase II deficiency or TREX1 deficiency
• an autoimmune disease an autoimmune disease
• SLE systemic lupus erythematosus
• Sjögren's syndrome or a cardiovascular disease.
• the disorder or condition is senescence or aging.
• the subject is a mammal. In some embodiments, the mammal is a human.
• the invention provides a compound suitable for the degradation of Stimulator of Interferon Genes (STING), wherein the chimeric compound has a formula S1-L-E, wherein S1 is a STING ligand; L is a linker; E is a E3 ubiquitin ligase ligand; and wherein the STING ligand binds to STING non-covalently, for use as a medicament.
• STING Stimulator of Interferon Genes
• the invention provides a compound suitable for the degradation of Stimulator of Interferon Genes (STING), wherein the chimeric compound has a formula S1-L-E, wherein S1 is a STING ligand; L is a linker; E is a E3 ubiquitin ligase ligand; and wherein the STING ligand binds to STING non-covalently, for the manufacture of a medicament for the treatment of a disease or condition.
• STING Stimulator of Interferon Genes
• the invention provides a method of treatment of a disease or condition in a subject comprising administering a compound suitable for the degradation of Stimulator of Interferon Genes (STING), wherein the chimeric compound has a formula S1-L-E, wherein S1 is a STING ligand; L is a linker; E is a E3 ubiquitin ligase ligand; and wherein the STING ligand binds to STING non-covalently.
• STING Stimulator of Interferon Genes
• the invention provides a use of a compound in the manufacture of a medicament for the treatment of a disease or condition, wherein the bifunctional compound is suitable for the degradation of Stimulator of Interferon Genes (STING), wherein the chimeric compound has a formula S1-L-E, wherein S1 is a STING ligand; L is a linker; E is a E3 ubiquitin ligase ligand; and wherein the STING ligand binds to STING non-covalently.
• STING Stimulator of Interferon Genes
• the invention provides a method of inhibiting signaling driven by constitutive activation of STING, the method comprising administration of a therapeutically effective amount of a compound suitable for the degradation of Stimulator of Interferon Genes (STING), wherein the chimeric compound has a formula S1-L-E, wherein S1 is a STING ligand; L is a linker; E is a E3 ubiquitin ligase ligand; and wherein the STING ligand does not form a covalent bond with STING.
• STING Stimulator of Interferon Genes
• S1 is a STING ligand.
• the STING ligand does not form a covalent bond with STING.
• the STING ligand does not exhibit STING agonist activity.
• Exhibiting STING agonist activity means inducing cytokine induction or inducing phospho-STING. Therefore, the STING ligand S1 does not induce cytokine induction nor induce phospho-STING.
• Any standard method known in the art may be used to determine STING agonist activity.
• STING agonist activity (or lack thereof) can be measured as described in Examples 48 and/or Example 51.
• cells may be incubated with a compound comprising a S1 STING ligand. The cells may be lysed and Western Blotting performed.
• Western blots may be probed for phospho-STING and/or phospho-TBK1.
• Cells incubated with STING ligands which are not agonists will not result in detectable phospho-STING or phospho-TBK1 using the methods outlined above and in Examples 48.
• human skin fibroblasts may be pretreated for 2hours with a serial dilution of a test compounds, for example a compound comprising an S1 ligand, prior to stimulation with a STING agonist (e.g. 300 nM GSK STING agonist).
• STING agonist e.g. 300 nM GSK STING agonist
• IFN-a/b may be measured in the supernatants using HEK-Blue IFN- ⁇ / ⁇ bioassay (Invivogen) to determine a half-maximal inhibitory concentration of a test compound.
• a compound comprising an S1 ligand having an IC50 value against STING indicates that the compound is not a STING agonist.
• STING degradation may be quantified using any standard method in the art. For example, STING degradation may be measured using Western Blot analysis as described in Example 11 followed by quantification of protein band signal intensity (e.g. densiometric analysis). Quantification of may be performed using any commonly available software, for example Image Lab software (Biorad, Version 6.0.1).
• incubating cells with 1 ⁇ M S1-L-E compound for 16 hours reduces relative STING protein abundance by at least 30%, at least 35%, at least 40%, at least 45%, 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% as compared to cells not treated with S1-L-E compound.
• incubating cells with 5 ⁇ M S1-L-E compound for 16 hours reduces relative STING protein abundance by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% as compared to cells not treated with S1-L-E compound.
• incubating cells with 7.5 ⁇ M S1-L-E compound for 16 hours reduces relative STING protein abundance by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% as compared to cells not treated with S1-L-E compound.
• incubating cells with 10 ⁇ M S1-L-E compound for 16 hours reduces relative STING protein abundance by at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% as compared to cells not treated with S1-L-E compound.
• the cells incubated with the S1-L-E compounds are selected from the following: H1650 cells, H596 cells, HaCat cells, HFF-1 cells, RAW264.7 cells, primary fibroblasts, primary SAVI patient fibroblasts, primary human skin fibroblasts, and THP-1 cells.
• R 9 and R 19 are each independently selected from an optionally substituted C 5-10 cycloalkyl, optionally substituted C 6-10 carboaryl, optionally substituted C 5-10 heteroaryl or optionally substituted C 5-10 heterocyclyl, wherein the optional substituents are independently selected from H, halogen, CF 3 , optionally substituted C 3-5 cycloalkyl, optionally substituted C 3-5 carbocyclyl, optionally substituted C 4-5 heterocyclyl, optionally substituted C 5 heteroaryl, optionally substituted C 1-4 alkyl and optionally substituted C 1-4 alkoxy, wherein said optionally substituted C 3-5 cycloalkyl, optionally substituted C 3-5 carbocyclyl, optionally substituted C 4-5 heterocyclyl, optionally substituted C 5 heteroaryl, optionally substituted C 1-4 alkyl and optionally substituted C 1-4 alkoxy is optionally substituted by one or more substituents independently selected from F, CF 3 , CI,
• R 9 and R 19 are each independently selected from an optionally substituted C 5-6 heteroaryl or optionally substituted C 5-6 heterocyclyl, wherein the optional substituents are independently selected from H, halogen, CF 3 , optionally substituted C 3-5 cycloalkyl, optionally substituted C 3-5 carbocyclyl, optionally substituted C 4-5 heterocyclyl, optionally substituted C 5 heteroaryl, optionally substituted C 1-4 alkyl, and optionally substituted C 1-4 alkoxy, wherein said optionally substituted C 3-5 cycloalkyl, optionally substituted C 3-5 carbocyclyl, optionally substituted C 4-5 heterocyclyl, optionally substituted C 5 heteroaryl, optionally substituted C 1-4 alkyl and optionally substituted C 1-4 alkoxy is optionally substituted by one or more substituents independently selected from F, CF 3 , CI, OH, CN, NH 2 , C 1-3 alkoxy, CO 2 (C 1-3
• R 9 and R 19 are each independently selected from an optionally substituted C 5-6 heteroaryl or optionally substituted C 5-6 heterocyclyl, wherein the optional substituents are independently selected from H, halogen, C 3-5 cycloalkyl, optionally substituted C 1-4 alkyl wherein said optionally substituted alkyl is optionally substituted by one or more substituents independently selected from halogen, halo(C 1-4 alkyl), OH, NH 2 , C 1-4 alkoxy, CO 2 H, CO 2 C 1-6 alkyl, N(C 1-6 alkyl) 2 , OCONH 2 and CONH 2 .
• R 9 or R 19 is an optionally substituted C 5-6 heteroaryl or optionally substituted C 5-6 heterocyclyl the optional substituent is selected from optionally substituted methyl, optionally substituted ethyl, optionally substituted propyl wherein the optional substituents are OH, NH 2 , halogen or C 1-4 alkoxy.
• R 9 or R 19 is an optionally substituted C 5-6 heteroaryl or optionally substituted C 5-6 heterocyclyl the optional substituent is selected from methyl and ethyl.
• formula (I) is of formula (I-2):
• R 1 and R 11 are each independently selected from H, halogen, OH, optionally substituted C 1-6 alkyl, optionally substituted C 1-6 alkoxy, optionally substituted CO 2 C 1-6 alkyl, and optionally substituted C 1-6 alkylamino wherein the optional substituents are independently selected from OH, -OP(O)(OH) 2 , C 1-4 alkoxy, NH 2 , NH(C 1-6 alkyl), N(C 1-6 alkyl) 2 , CO 2 (C 1-6 alkyl) and halogen.
• R 1 or R 11 are optionally substituted C 1-6 alkyl
• the optional substituents are selected from OH, F, Br, methoxy and ethoxy.
• R 1 or R 11 are optionally substituted C 1-6 alkyl, they are optionally substituted methyl or optionally substituted ethyl. In further embodiments, they are optionally substituted methyl. In further embodiments, they are unsubstituted methyl.
• R 1 or R 11 are halo, in some embodiments they are F, Br or Cl.
• R 1 and R 11 are independently selected from H, OH, F and methyl.
• R 1 is identical to R 11 .
• R 1 and R 11 are H.
• R 2 and R 12 are H.
• the optional substituents are selected from OH, -O-P(O)(OH) 2 , methoxy, ethoxy, N(CH 3 ) 2 , NH(CH 3 ), NH 2 , CO 2 (CH 3 ) and halogen.
• R 3 is identical to R 13 .
• R 4 and R 14 are each independently selected from H, halogen, OH, optionally substituted C 1-6 alkyl, optionally substituted C 1-6 alkoxy, optionally substituted CO 2 C 1-6 alkyl, and optionally substituted C 1-6 alkylamino wherein the optional substituents are selected from OH, -O-P(O)(OH) 2 , C 1-4 alkoxy, N(C 1-6 alkyl) 2 , NH(C 1-6 alkyl), NH 2 , CO 2 (C 1-6 alkyl) and halogen.
• R 4 or R 14 are optionally substituted C 1-6 alkyl
• the optional substituents are selected from OH, F, Br, methoxy and ethoxy.
• R 4 or R 14 are optionally substituted C 1-6 alkyl, they are optionally substituted methyl or optionally substituted ethyl. In further embodiments, they are optionally substituted methyl. In further embodiments they are unsubstituted methyl.
• R 4 or R 14 are halo, in some embodiments they are F, Br or Cl.
• R 4 and R 14 are each independently selected from H, OH, F or methyl.
• R 4 is identical to R 14 .
• R 5 and R 15 are each independently selected from C 1-4 alkyl or C 1-4 alkoxy.
• R 5 and R 15 are each independently selected from H, cyclopropyl and C 1-4 alkyl.
• R 5 and R 15 are both H.
• R 5 and R 15 are both cyclopropyl.
• R 5 or R 15 are each independently C 1-4 alkyl they are methyl, ethyl, propyl or butyl. In some embodiments they are methyl or ethyl. In further embodiments they are methyl.
• R 5 is identical to R 15 .
• R 5 and R 15 are methyl.
• R 6 and R 16 are each independently selected from H, halogen or optionally substituted C 1-4 alkyl or C 1-4 alkoxy wherein the optional substituents are independently selected from halogen, halo(C 1-4 alkyl), OH, and C 1-4 alkoxy.
• R 6 or R 16 are optionally substituted C 1-6 alkyl
• the optional substituents are selected from OH, F, Br, methoxy and ethoxy.
• R 6 or R 16 are optionally substituted C 1-6 alkyl, they are optionally substituted methyl or optionally substituted ethyl. In further embodiments, they are optionally substituted methyl. In further embodiments, they are unsubstituted methyl.
• R 6 or R 16 are halo, in some embodiments they are F, Br or Cl.
• R 6 and R 16 are each independently selected from H, OCH 3 , halogen or C 1-3 alkyl.
• R 6 and R 16 are each independently selected from H, OCH 3 , F or methyl.
• R 6 is identical to R 16 .
• R 6 and R 16 are H.
• R 7 and R 17 are each independently selected from optionally substituted C 1-4 alkyl wherein the optional substituents are independently selected from halogen, -halo(C 1-4 alkyl), OH, and C 1-4 alkoxy.
• R 7 or R 17 are each independently optionally substituted C 1-4 alkyl it is an optionally substituted methyl, ethyl, propyl or butyl.
• R 7 or R 17 are each independently selected from optionally substituted C 1-4 alkyl the optional substituents are selected from OH, halogen, methoxy and ethoxy.
• R 7 and R 17 are each independently optionally substituted C 1-4 alkyl, in some embodiments they are methyl or ethyl.
• R 7 is identical to R 17 .
• R 7 and R 17 are both C 1-4 alkyl.
• R 7 and R 17 are ethyl.
• R 8 and R 18 are each independently selected from H, and C 1-4 alkyl.
• R 8 and R 18 are C 1-4 alkyl they are selected from methyl, ethyl, propyl or butyl.
• R 8 and R 18 are methyl, ethyl or H. In further embodiments R 8 and R 18 are methyl or H.
• R 8 is identical R 18 .
• R 8 and R 18 are H.
• R 8 and R 18 are methyl.
• R 8 and R 18 are ethyl.
• R B1 and R B2 are each independently selected from (-CH 2 -) 1-2 or a bond.
• R B1 or R B2 are a bond, this is a bond connected directly from the N to B'.
• R B1 or R B2 are (-CH 2 -) 1-2 they can each independently be -CH 2 - or -CH 2 CH 2 -.
• R B1 is a bond and R B2 is -CH 2 - or -CH 2 CH 2 -. In some embodiments R B1 is a bond and R B2 is -CH 2 - or -CH 2 CH 2 -. In other embodiments R B2 is a bond and R B1 is -CH 2 -. In other embodiments R B1 is a bond and R B2 is -CH 2 -.
• R B1 and R B2 are both a bond.
• R B1 and R B2 are both -CH 2 - or both -CH 2 -CH 2 - or R B1 is -CH 2 - and R B2 is -CH 2 -CH 2 -In some embodiments R B1 is identical to R B2 .
• R B1 and R B2 are both -CH 2 -.
• R B1 and R B2 are both -CH 2 CH 2 -.
• B' is CH, methyl, ethyl, propyl, butyl or pentyl. In further embodiments B is CH, methyl or ethyl.
• B is CH or C 1-2 alkyl and B' together with R B1 and R B2 form a C 3-8 alkyl chain.
• B is CH or C 1-2 alkyl and B' together with R B1 and R B2 form a C 5 alkyl chain.
• m is 0.
• n 1
• m is 0 and therefore B' is bonded directly to O and B' is CH or C 1-2 alkyl.
• m is 0 and therefore B' is bonded directly to O and B' together with R B1 and R B2 form a C 3-8 alkyl chain. In further embodiments m is 0 and therefore B' is bonded directly to O and B' together with R B1 and R B2 form a C 5 alkyl chain.
• B' is of the formula:
• R B1 and R B2 are each -CH 2 -CH 2 - and together with B' have the following structure: wherein the wavy line next to L represents the attachment to the linker and the wavy lines next to the alkyl groups represent the connection to the N atoms in the S1 structure.
• R 1 is identical to R 11
• R 2 is identical to R 12
• R 3 is identical to R 13
• R 4 is identical to R 14
• R B1 is identical to R B2
• R 8 is identical to R 18 and R 9 is identical to R 19
• R 1 is identical to R 11
• R 2 is identical to R 12
• R 3 is identical to R 13
• R 4 is identical to R 14
• R 5 is identical to R 15
• R B1 is identical to R B2
• R 8 is identical to R 18
• R 6 is identical to R 16 and R 7 is identical to R 17 . Therefore, in some embodiments S1 is symmetrical.
• R 4 , R 14 , R 2 , R 12 , R 1 and R 11 are each independently selected from H, OH, OCH 3 , halogen or C 1-3 alkyl.
• R 7 , R 8 , R 17 and R 18 are C 1-4 alkyl.
• compound (I) is of the Formula (I-2b): or a tautomer, or a pharmaceutically acceptable salt thereof wherein B' is a C 1-5 alkyl and is the connection point to the Linker.
• compound of formula (I) is of the formula (I-2c) or (I-2d):
• the E3 ubiquitin ligase ligand is a Cereblon (CRBN) E3 ubiquitin ligase ligand. In some embodiments the E3 ubiquitin ligase ligand is a von Hippel-Lindau (VHL) E3 ubiquitin ligase ligand. In some embodiments the E3 ubiquitin ligase ligand is an Inhibitor of Apoptosis (IAP) E3 ubiquitin ligase ligand.
• CBN Cereblon
• VHL von Hippel-Lindau
• IAP Inhibitor of Apoptosis
• E has a structure according to Formula E1: wherein
• R E1A is optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, or optionally substituted C 5-10 heterocyclyl
• the optional substituents are selected from methyl, ethyl, OH, F or Cl.
• R E1A is optionally substituted C 3-7 cycloalkyl, in some embodiments it is cyclopropyl, cyclobutyl or cyclopentyl.
• R E1A is C 1-6 alkyl, in some embodiments it is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl or tert-butyl.
• R E1A is C 1-6 alkyl, in some embodiments it is a branched C 1-4 alkyl.
• R E1A is optionally substituted C 1-6 alkyl
• R E1A is a branched butyl group.
• R E1A is tert-butyl.
• R E1B when R E1B is OR, NHR or NRR', R and R' are independently selected from methyl, ethyl, propyl, cyclopropyl or cyclobutyl. In some embodiments when R E1B is OR it is O-methyl or O-ethyl. In some embodiments when R E1B is NHR or NRR', it is NHCH 3 , NHCH 2 CH 3 , N(CH 3 )(CH 3 ), N(CH 2 CH 3 )(CH 3 ) or N(CH 2 CH 3 )(CH 2 CH 3 ).
• R E1B is H.
• R E1B is OH.
• R E1C is H.
• R E1C is C 1-3 alkyl. In further embodiments R E1C is methyl.
• Ring B is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring B is an optionally substituted C 6-10 carboaryl, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring B is an optionally substituted C 5-10 heteroaryl group, in some embodiments it is an optionally substituted C 5-6 heteroaryl group which comprises 1 or 2 heteroatoms selected from O, S and N. In some embodiments the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• Ring B is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, in some embodiments the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• Ring B when Ring B is an optionally substituted C 6-10 carboaryl it is an optionally substituted phenyl wherein the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• Ring B is phenyl
• Ring B is phenyl linked in positions 1 and 4.
• Ring C is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring C is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group
• the optional substituents are selected from methyl, ethyl, OH, F or Cl. In one embodiment there are 1 or 2 optional substituents. In some embodiments the optional substituent is methyl.
• Ring C when Ring C is an optionally substituted C 6-10 carboaryl it is an optionally substituted phenyl wherein the optional substituents are selected from methyl, ethyl, OH, F or Cl. In some embodiments Ring C is an unsubstituted phenyl.
• Ring C is an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH. In some embodiments the optional substituents are selected from methyl, ethyl, OH, F or Cl.
• Ring C When Ring C is an optionally substituted C 5-10 heteroaryl group, in some embodiments it is an optionally substituted C 5-6 heteroaryl group. In some embodiments Ring C has 1 or 2 heteroatoms selected from O, S and N. In some embodiments Ring C is selected from azolyl, pyridiyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isoxazinyl, thiazolyl, isothiazolyl, imidazolyl, pyrazoyl, pyridazinyl, pyrimidinyl or pyrazinyl. In some embodiments Ring C has 1 or 2 heteroatoms selected from S and N.
• Ring C is an optionally substituted C 5-10 heteroaryl group, in some embodiments it is an optionally substituted C 5 heteroaryl group. In some embodiments Ring C has two heteroatoms one being N and the other being S. In some embodiments the optional substituents are methyl. In some embodiments there is one methyl substituent on the C 5 heteroaryl.
• Ring C is a 1,3-thiazole optionally substituted by methyl. In further embodiments Ring C is 4-methyl-1,3-thiazol-5-yl.
• E has the formula E1 wherein R E1A is a branched C 1-4 alkyl, R E1B is selected from OH, O-methyl or O-ethyl, R E1C is H or methyl, Ring B is an optionally substituted phenyl wherein the optional substituents are selected from methyl, ethyl, F, Cl and OH and Ring C is an optionally substituted C 5-10 heteroaryl group wherein the optional substituents are selected from methyl, ethyl, OH, F or Cl.
• E has the formula E1 wherein R E1A is tert-butyl, R E1B is OH, Ring B is phenyl and Ring C is a 1,3-thiazole substituted by methyl.
• E has the following structure E1-1: wherein represents attachment point to L.
• E has the following structure E1-2: wherein represents attachment point to L.
• E has a structure according to Formula E2: wherein
• R when R is C 1-6 alkyl it is methyl, ethyl or propyl. In some embodiments when R is C 1-6 alkenyl it is ethenyl or propenyl. In some embodiments when R is C 3-7 cycloalkyl it is cyclopropyl or cyclobutyl. In some embodiments R is H, OH or methyl. In some embodiments R is H. In some embodiments when R is C 1-6 alkoxy it is methoxy or ethoxy. In further embodiments when R is C 1-6 alkoxy it is OMe.
• R E2B when R E2B is C 1-6 alkyl it is methyl, ethyl or propyl. In some embodiments R E2B is selected from H, F, Cl or methyl. In some embodiments R E2B is H or F. In some embodiments R E2B is H.
• R E2C when R E2C is C 1-6 alkyl it is methyl, ethyl or propyl. In some embodiments R E2C is selected from H, OH, or methyl. In further embodiments R E2C is H.
• R E2D is selected from OH, halogen, C 1-6 alkoxy, and C 1-6 alkyl. In some embodiments, R E2D is C 1-6 alkoxy.
• one of Q 1 , Q 2 , Q 3 , and Q 4 when one of Q 1 , Q 2 , Q 3 , and Q 4 is CR E2A , the remaining are CH or CR E2D . In further embodiments one of Q 1 , Q 2 , Q 3 , and Q 4 is CR E2A , and the remaining are CH. In some embodiments, one of Q 1 , Q 2 , Q 3 , and Q 4 is CR E2A , another is CR E2D , and the remaining are CH.
• one of Q 1 , Q 2 , Q 3 , and Q 4 is CR E2A , another is COMe, and the remaining are CH.
• Q 4 is CR E2A
• Q 1 , Q 2 and Q 3 are CH or CR E2D
• Q 4 is CR E2A
• Q 1 , Q 2 and Q 3 are CH or CR E2D
• Q 4 is CR E2A
• Q 1 , Q 2 and Q 3 are CH.
• R E2A is -O- or -NR- wherein R is H, OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 cycloalkyl, C 4-7 heterocyclyl.
• R E2A is -NR and R is C 1-6 alkyl, R can be methyl or ethyl.
• R E2A is -NR and R is C 3-7 cycloalkyl R can be cyclopropyl.
• R E2A is -NR, R is H.
• R E2A is -NR-. In further embodiments R E2A is -NH-. Therefore, in some embodiments E is joined to R E2A via an -N(H)- bond.
• R E2A is a bond
• Q 1 , Q 2 and Q 3 are CH and Q 4 is CR E2A
• R E2A is a bond
• R E2B is H
• R E2C is H.
• E has the following structure E2-1: wherein represents attachment point to L.
• E has the following structure E2-2: wherein represents attachment point to L.
• E has a structure according to Formula E3 wherein
• R E3A is optionally substituted C 5-10 cycloalkyl
• optionally substituted C 6-10 carboaryl the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• R E3A is an optionally substituted C 5-10 cycloalkyl it is selected from cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl, cyclooctyl, cyclononayl, cyclodecyl, decalinyl and 2,3,3a,4,5,6,7,7a-octahydro-1H-indenyl.
• R E3A is an optionally substituted C 6-10 carboaryl in some embodiments it is a single ring selected from phenyl, naphthyl and azulyl.
• R E3A is an optionally substituted C 6-10 carboaryl in some embodiments it is fused rings, at least one of which is an aromatic ring, selected from indanyl (e.g. 2,3-dihydro-1H-indene), indenyl, isoindenyl (C 9 ) and tetralin-1-yl (e.g. 1,2,3,4-tetrahydronaphthalene).
• R E3A is an unsubstituted tetralin. In some embodiments, R E3A is ( 1R )-1,2,3,4-tetrahydronaphthalen-1-yl.
• R E3A is an unsubstituted tetralin-1-yl which has the following structure:
• R E3B is C 3-7 cycloalkyl.
• R E3B is C 3-7 cycloalkyl it is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl methylcyclohexyl.
• R E3B is cyclopentyl, cyclohexyl or cycloheptyl.
• R E3B is cyclohexyl
• R E3B is cyclohexyl and R E3A is an unsubstituted tetralin-1-yl.
• E has the structure E3-1: wherein represents attachment point to L.
• E has a structure according to Formula E4 wherein
• R E4 is C 3-7 cycloalkyl in some embodiments it is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl methylcyclohexyl.
• R E4 is cyclopentyl, cyclohexyl or cycloheptyl.
• R E4 is a C 3-7 cycloalkyl.
• R E4 is a C 5-6 cycloalkyl.
• R E4 is cyclohexyl
• Ring D is a C 5-7 heteroaryl.
• Ring D is a C 5-7 heteroaryl in some embodiments it contains 1 to 4 heteroatoms selected from O, N and S. In some embodiments it is a 5 or 6 membered heteroaryl with 1 or 2 heteroatoms. In some embodiments it is a 5 membered heteroaryl with 2 heteroatoms. In some embodiments it is a 5 membered heteroaryl with 2 heteroatoms one being N and the other being S.
• Ring D is an N containing C 5-7 heteroaryl group. In some embodiments Ring D a C 5-7 heteroaryl group containing one N atom and one S atom.
• Ring D is a C 5-7 heteroaryl in some embodiments it is a pyrrolyl, pyridyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isoxainyl, oxadiazolyl, oxatirazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl or tetrazolyl.
• Ring E is a C 6 heteroaryl containing 1 heteroatom selected from O, N or S.
• Ring E is C 6-10 carboaryl.
• Ring E is C 6-10 carboaryl in some embodiments it is phenyl.
• R E4 is C 5-6 cycloalkyl
• Ring D is a C 5-7 heteroaryl group containing one S and one N
• Ring E is phenyl.
• E has the following structure E4-1: wherein represents attachment point to L.
• E has a structure according to E5:
• Ring F2 is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring F2 is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from methyl, ethyl, propyl, OH or halogen. In some embodiments the optional substituents are selected from methyl, ethyl, OH, F or Cl. In one embodiment there are 1 or 2 optional substituents. In some embodiments the optional substituent is methyl.
• Ring F2 when Ring F2 is an optionally substituted C 6-10 carboaryl it is an optionally substituted phenyl wherein the optional substituents are selected from methyl, ethyl, propyl, OH, F or Cl. In some embodiments Ring F2 is an unsubstituted phenyl.
• Ring F2 is an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH. In some embodiments the optional substituents are selected from methyl, ethyl, OH, F or Cl.
• Ring F2 is an optionally substituted C 5-6 heteroaryl group, wherein the optional substituents are selected from methyl, ethyl, propyl, halogen or OH.
• Ring F2 When Ring F2 is an optionally substituted C 5-10 heteroaryl group, in some embodiments it is an optionally substituted C 5-6 heteroaryl group. In some embodiments Ring F2 has 1 or 2 heteroatoms selected from O, S and N. In some embodiments Ring F2 is selected from azolyl, pyridiyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isoxazinyl, thiazolyl, isothiazolyl, imidazolyl, pyrazoyl, pyridazinyl, pyrimidinyl or pyrazinyl. In some embodiments Ring F2 has 1 or 2 heteroatoms selected from S and N.
• Ring F2 is an optionally substituted C 5-10 heteroaryl group, in some embodiments it is an optionally substituted C 5 heteroaryl group. In some embodiments Ring F2 has two heteroatoms one being N and the other being S. In some embodiments the optional substituents are methyl. In some embodiments there is one methyl substituent on the C 5 heteroaryl.
• Ring F2 is a 1,3-thiazole optionally substituted by methyl. In further embodiments Ring F2 is 4-methyl-1,3-thiazol-5-yl.
• Ring F2 is
• Ring F1 is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring F1 is an optionally substituted C 5-10 heteroaryl group it is an optionally substituted C 5-6 heteroaryl group which comprises 1 or 2 heteroatoms selected from O, S and N.
• the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• Ring F1 is an optionally substituted C 6-10 carboaryl, wherein the optional substituents are selected from C 1-6 alkyl, halogen or OH.
• Ring F1 is an optionally substituted C 6-10 carboaryl, or an optionally substituted C 5-10 heteroaryl group, in some embodiments the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• Ring F1 is an optionally substituted C 6-10 carboaryl it is an optionally substituted phenyl wherein the optional substituents are selected from methyl, ethyl, F, Cl and OH.
• R E51 is H, OH, OR or NH 2 , wherein R is selected from C 1-6 alkyl or C 3-7 cycloalkyl. In some embodiments when R E51 is OR, R is methyl, ethyl or cyclopropyl. In some embodiments R E51 is H, OH or OMe. In further embodiments R E51 is OH.
• R 53 is optionally substituted with F, Cl, Br, CN, OH or methyl. In some embodiments R 53 is optionally substituted with F or Cl. In further embodiments R 53 is substituted with F.
• R E54 is H. In some embodiment R E54 is methyl, ethyl, propyl or butyl.
• E5 is of the following formula E5-1: wherein ' represents attachment point to L.
• E has a structure according to Formula E6: wherein
• one of Q 5 , Q 6 , Q 7 , Q 8 , and Q 9 is CR E6A , and no more than one of Q 5 , Q 6 , Q 7 , or Q 8 are N.
• one of Q 5 , Q 6 , Q 7 , Q 8 , and Q 9 is CR E6A , and the remaining are CH or CR E6D .
• R E6D is selected from halogen, C 1-6 alkoxy, and C 1-6 alkyl.
• R E6D is selected from C 1-6 alkoxy, and C 1-6 alkyl.
• R E6D is selected from F, Cl, Br, and OMe
• Q 6 is CR E2A .
• R E6A is a bond, or -NR- wherein R is H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 cycloalkyl, C 4-7 heterocyclyl. In some embodiments, R E6A is a bond. In some embodiments, R E6A is -NR-wherein R is H, OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 cycloalkyl, C 4-7 heterocyclyl. In some embodiments R E6A is -NR- wherein R is H or C 1-6 alkyl. In some embodiments R E6A is -NH-.
• R E6B is H, F, Cl, or Br. In some embodiments R E6B is C 1-3 alkyl. In some embodiments, R E6B is H or fluorine. In some embodiments R E6B is H.
• R E6C is selected from H, OH, and C 1-6 alkyl. In some embodiments R E6C is H.
• R E6D is selected from OH, halogen, C 1-6 alkoxy, and C 1-6 alkyl. In some embodiments, R E2D is C 1-6 alkoxy.
• E6 is formula E6-1 and has the following structure: wherein represents attachment point to L
• E6 is formula E6-2 and has the following structure: wherein represents attachment point to L
• E6 is formula E6-3 and has the following structure: wherein represents attachment point to L
• E is selected from one of the following structures: E Structure E Structure E1-1 E4-1 E1-2 E5-1 E2-1 E6-1 E2-2 E6-2 E3-1 E6-3
• L is a linker.
• L is a linker which connects S1 to E.
• L connects the STING ligand to the E3 ubiquitin ligase ligand via a chain of atoms having 10-40 atoms in shortest length. In further embodiments L connects the STING ligand to the E3 ubiquitin ligase ligand via a chain of atoms having 15-36 atoms in shortest length.
• L has the following structure: *-L A -[X] X -L B -* wherein
• L1 when L1 is C 1-6 alkyl it is methyl, ethyl or propyl. In some embodiments L1 is methyl.
• a is 0. In some embodiments a is 1.
• L1 is methyl and a is 1.
• b is 0 to 6. In some embodiments b is 1 to 5. In some embodiments b is 2 to 5. In some embodiments b is 2, 3, 4 or 5.
• L2 when L2 is C 1-6 alkyl it is methyl, ethyl or propyl. In some embodiments L1 is methyl or ethyl. In some embodiments L2 is ethyl.
• c is 0. In some embodiments c is 1.
• L2 is ethyl and c is 1.
• d is 0. In some embodiments d is 1.
• e 0.
• e is 1.
• the C 6 heterocyclyl is selected from piperidine, piperazine or morpholine.
• the C 6 heteroaryl is selected from pyridine, pyrimidine or pyrazine.
• f is 0 to 5. In some embodiments f is 0. In some embodiments f is 1. In some embodiments f is 2. In some embodiments f is 3. In some embodiments f is 4. In some embodiments f is 5.
• g 0.
• h 0.
• L7 is C 3-6 heterocyclyl. In some embodiments L7 is piperidinyl or piperazinyl.
• L7 is -NH- or C 3-6 heterocyclyl, and i is 1 or 2.
• L7 is -NH-, and i is 1.
• i is 0. In some embodiments i is 1. In some embodiments i is 2.
• linker L is selected from one of the following structures: L Structure LK1 LK2 LK3 LK4 LK5 LK6 LK7 LK8 LK9 LK10 LK11 LK12 LK13 LK14 LK15 LK16 LK17 LK18 LK19 LK20 LK21 LK22 LK23 LK24 LK25 LK26
• S1-L-E is selected from one of the following formulae: or tautomers thereof, wherein the substituents R 1 , R 2 , R 3 , R 4 , R 8 , R 9 , R B1 , B', Linker, R B2 , R 11 , R 12 , R 13 , R 14 , R 18 , R 19 R E1A , R E1B , R E1C , Rings B, C, D, E, F1, F2, Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 , Q 8 , Q 9 , Z E2 , X E2b , R E2B , X E2a , R E2C , R E2D , Y E2 , X E6a , Y E6 , R E6B , R E6C , R E3A , R E3B , R E4 , R E51 , R E
• R 9 is: and R 19 is:
• R 9 and R 19 are each:
• S1-L-E is selected from one of the following formulae:
• the compound is selected from one of the following compounds 1-38 in Table 1.
• Table 1 Example number Structure IUPAC Name 1 1-[3-( ⁇ N-2-[2-(2- ⁇ 2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-4-isoindolinylamino]ethoxy ⁇ ethox y)ethoxy]ethylcarbamoyl ⁇ metho xy)-5- ⁇ 5-carbamoyl-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazol-1-yl ⁇ pentyl]-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazole-5-carboxamide 2 N- ⁇ 1-[3-( ⁇ N-2-[2-(2-
• Step 1' To a stirred suspension of 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (4 g, 25.95 mmol) in anhydrous DCM (80 mL) at 0 °C were added oxalyl chloride (2.67 mL, 31.1 mmol) and DMF (0.2 mL, 2.59 mmol) dropwise. The reaction mixture was stirred at 0 °C for 15 min and then allowed to warm to rt (after 15 min the slurry turned into a light yellow solution). The reaction mixture was stirred for 1 h at rt.
• Step 2' 1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl chloride (4.48 g, 25.95 mmol) was placed under nitrogen and dry acetone (66 mL) was added. The reaction mixture was cooled down to 0 °C and potassium thiocyanate (3.03 g, 31.1 mmol) was added portionwise. The reaction mixture was stirred for 15 min at 0 °C and then for 1 h at rt. 50 mL of isohexane was added and the reaction mixture was concentrated in vacuo to dryness. To the black residue 100 mL of isohexane were added and the resulting dark brown solid was collected by filtration and washed with isohexane twice.
• Step 1 To a stirred solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (538 mg, 2.78 mmol) in acetone (7.0 mL) was added a solution of K 2 CO 3 (770 mg, 5.57 mmol) in water (7.0 mL). The mixture was cooled to 0 °C. Allylchloroformate (0.30 mL, 2.78 mmol) was added. The reaction mixture was stirred at rt for several hours until consumption of starting material was observed by TLC (DCM-MeOH 9:1). Acetone was removed under reduced pressure. DCM was added and the aqueous phase was extracted with DCM (3x 8 mL).
• Step 2 To a stirred solution of the product of Step 1 (300 mg, 1.08 mmol) in dry DCM (4.3 mL) at 0 °C were added TsCI (247 mg, 1.30 mmol), DMAP (13 mg, 0.108 mmol) and triethylamine (0.23 mL, 1.62 mmol). The reaction mixture was stirred at rt overnight. Saturated aqueous sodium bicarbonate solution was added and the organic phase was separated. The aqueous phase was further extracted with DCM (2x 20 mL). The combined organic phases were concentrated under reduced pressure.
• Step 3 A mixture of 2-(2- ⁇ 2-[2-(allyloxycarbonylamino)ethoxy]ethoxy ⁇ ethoxy)ethyl 4-methylbenzenesulfonate (26 mg, 0.0602 mmol), tert-butyl ((S)-1-(((S)-1-cyclohexyl-2-((S)-2-(4-(3-hydroxybenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (30 mg, 0.0501 mmol; available e.g. from Tocris Bioscience, cat.no.
• Step 4 A stirred solution of the crude product of Step 3 (43 mg, 0.0501 mmol) and phenylsilane (0.019 mL, 0.154 mmol) in dry DCM (0.60 mL) was degassed and purged five times with nitrogen. Then, tetrakis(triphenylphosphine)palladium(0) (5.8 mg, 0.0050 mmol) was added and the reaction mixture was stirred at rt for 50 min. The reaction mixture was concentrated under reduced pressure, then re-dissolved in a minimum amount of MeOH and loaded to an SCX cartridge (sorbent bed weight 0.5 g). The cartridge was flushed with MeOH (3 CV). The product was eluted with 2M NH 3 in MeOH (3 CV).
• Step 1 Methyl 4-bromo-2-methoxybenzoate (250 mg, 1.02 mmol), tris(dibenzylideneacetone) dipalladium (0) (93 mg, 0.102 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (64 mg, 0.102 mmol), cesium carbonate (997 mg, 3.06 mmol) and tert-butyl 2-(2-aminoethoxy)ethylcarbamate (229 mg, 1.12 mmol) were placed under nitrogen and suspended in anhydrous 1,4-dioxane (2.6 mL). The reaction mixture was purged with nitrogen for 5 min and then heated at 105 °C overnight.
• reaction mixture was filtered through a celite cartridge (2.5 g), eluting with EtOAc.
• the filtrate was washed with brine (25 mL), passed through a hydrophobic frit, and concentrated under reduced pressure.
• the resulting material was purified via flash column chromatography (SiO 2 ; 5-95% EtOAc in isohexane), affording methyl 4-(2- ⁇ 2-[tert-butyl(oxycarbonylamino)]ethoxy ⁇ ethylamino)-2-anisate (318 mg, 85 %) as a yellow gum.
• Step 2 To a solution of methyl 4-(2- ⁇ 2-[tert-butyl(oxycarbonylamino)]ethoxy ⁇ ethylamino)-2-anisate (318 mg, 0.863 mmol) in methanol (3 mL) was added sodium hydroxide (345 mg, 8.63 mmol) in H 2 O (2 mL). The reaction was left to stir at 40 °C for 3 h. The pH was adjusted to pH 2 with 1 M HCl, and the mixture diluted with DCM (15 mL).
• Step 3 To a stirred solution of 4-(2- ⁇ 2-[tert-butyl(oxycarbonylamino)]ethoxy ⁇ ethylamino)-2-anisic acid (276 mg, 0.779 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (385 mg, 2.34 mmol) in dry DMF (6.8 mL) was added DIPEA (0.54 mL, 3.10 mmol) followed by EDC (149 mg, 0.779 mmol) and HATU (444 mg, 1.17 mmol). The reaction mixture was stirred at rt for 16 h. Saturated aq NH 4 Cl (100 mL) was added.
• Step 4 To a solution of 2- ⁇ 2-[3-methoxy-4-(N-2,6-dioxo-3-piperidylcarbamoyl)phenylamino]ethoxy ⁇ ethyl 2-methyl-2-propanecarbamate (66 mg, 0.142 mmol) in DCM (1.6 mL) was added TFA (0.72 mL). The mixture was stirred at rt for 30 min and then concentrated under reduced pressure to give the TFA salt of Intermediate L-4 (84 mg, 100 %) as a colourless oil.
• Step 1 2-Chloro-4-iodobenzoic acid (250 mg, 0.867 mmol), tris(dibenzylideneacetone) dipalladium (0) (79 mg, 0.087 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (54 mg, 0. 0.087 mmol), cesium carbonate (848 mg, 2.60 mmol) and tert-butyl [2-(piperidin-4-yl)ethyl]carbamate (218 mg, 0.954 mmol) were placed under nitrogen and suspended in anhydrous 1,4-dioxane (2.6 mL).
• Step 2 To a stirred solution of 4-(4- ⁇ 2-[tert-butyl(oxycarbonylamino)]ethyl ⁇ -1-piperidyl)-2-chlorobenzoic acid (32 mg, 0.084 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (42 mg, 0.254 mmol) in dry DMF (0.68 mL) was added DIPEA (0.059 mL, 0.339 mmol) followed by EDC (16 mg, 0.083 mmol) and HATU (48 mg, 0.127 mmol). The reaction mixture was stirred at rt for 30 min. Saturated aq NH 4 Cl (10 mL) was added.
• Step 3 The product of Step 2 (9.6 mg, 0.019 mmol) was dissolved DCM (0.23 mL) and TFA (0.8 mL). The mixture was stirred at rt for 2 h and then concentrated under reduced pressure to give the TFA salt of Intermediate L-7 (12.1 mg, 100 %) as a colourless oil.
• Step 1 To a suspension of 4-fluoro-3-nitrobenzamide (50 mg, 0.272 mmol) and 1-Boc-1,4-diaminobutane (51 mg, 0.272 mmol) in MeCN (1.4 mL) was added DIPEA (0.14 mL, 0.804 mmol). The reaction mixture was stirred at 80 °C for 6 h. The reaction mixture was concentrated under reduced pressure, affording 104 mg of crude tert -butyl (4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (96 mg, 0.272 mmol, 100 %) as a yellow solid.
• Step 2 To a stirred suspension of tert -butyl (4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (96 mg, 0.272 mmol) in DCM (2.2 mL) was added TFA (0.54 mL) dropwise. The reaction mixture was stirred at rt for 4 h (slurry became a solution after TFA addition, but the compound precipitated again). The reaction mixture was concentrated under reduced pressure, affording the product as the TFA salt that was taken to the next step without further purification.
• Step 3 To a stirred solution of 4-(4-aminobutylamino)-3-nitro-benzamide;2,2,2-trifluoroacetic acid (796 mg, 2.17 mmol) in DMF (11 mL) were added methyl 4-fluoro-3-nitrobenzoate (433 mg, 2.17 mmol) and DIPEA (1.9 mL, 10.9 mmol). The reaction mixture was stirred at 70 °C for 6 h and then cooled down to rt and poured into ice-cold water.
• Step 4 Methyl 4-((4-((4-carbamoyl-2-nitrophenyl)amino)butyl)amino)-3-nitrobenzoate (300 mg, 0.695 mmol) was suspended in 1-methyl-2-pyrrolidinone (36 mL) and MeOH (2.2 mL). The reaction was vac / filled with nitrogen three times. Then, palladium on carbon (74 mg, 0.695 mmol) was added under nitrogen and the reaction mixture was vac / filled with hydrogen three times leaving a balloon of hydrogen. The reaction mixture was stirred at rt overnight. The reaction mixture was filtered through celite, eluting with MeOH. The filtrate was concentrated under reduced pressure.
• Step 7 To a stirred solution of methyl 3-amino-4-((4-((2-amino-4-carbamoylphenyl)amino)butyl)amino)benzoate (50 mg, 0.135 mmol) in DMF (0.67 mL) at 0 °C was added 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (Intermediate 3; 53 mg, 0.269 mmol) dropwise. The reaction mixture was stirred at 0 °C for 20 min, and an LCMS sample was taken to confirm the formation of the thiourea intermediate.
• Step 8 To a stirred solution of methyl 1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)butyl)-2-(1 -ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazole-5-carboxylate (516 mg, 0.744 mmol) in MeOH (3.0 mL) and THF (3.0 mL) was added 1M aq LiOH (1.5 mL, 7.44 mmol) dropwise. The reaction mixture was stirred at rt for 48 h.
• Step 9 To a stirred solution of 1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)butyl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazole-5-carboxylic acid (50 mg, 0.0736 mmol) and N-Boc-1,3-diaminopropane (15 mg, 0.0883 mmol) in DMSO (2.0 mL) was added DIPEA (0.077 mL, 0.442 mmol).
• Step 10 To a stirred solution/suspension of tert-butyl (3-(1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)butyl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazole-5-carboxamido)propyl)carbamate (36 mg, 0.0388 mmol) in DCM (7.1 mL) at rt was added TFA (0.71 mL) dropwise. The reaction mixture was stirred at rt for 1 h.
• Step 11 To N-(3-aminopropyl)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)butyl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazole-5-carboxamide TFA salt (42 mg, 0.0388 mmol) in DMF (0.40 mL) was added triethylamine (0.032 mL, 0.233 mmol).
• Step 2 To a stirred solution of ethyl [2-cyano-1-(cyanomethyl)ethoxy]acetate (474 mg, 2.42 mmol) in ethanol (24 mL) was added hydrochloric acid (5 M solution in water, 1.4 mL, 7.0 mmol). The reaction mixture was vac / filled with nitrogen three times. Then, platinum (IV) oxide, Adam's catalyst (110 mg, 0.483 mmol) was added under N 2 and the reaction mixture was vac / filled with hydrogen three times. The reaction mixture was stirred at rt under 1 atm of hydrogen. Consumption of starting material was monitored by TLC.
• Step 3 To a stirred solution of 4-fluoro-3-nitrobenzamide (891 mg, 4.84 mmol) and ethyl [3-amino-1-(2-aminoethyl)propoxy]acetate dihydrochloride (670 mg, 2.42 mmol) in isopropanol (12 mL) was added DIPEA (4.2 mL, 24.1 mmol). The reaction mixture was stirred at 90 °C. After 1 h, the reaction mixture turned orange and a solid precipitate was observed.
• Step 4 A stirred solution of ethyl ⁇ 3-(4-carbamoyl-2-nitrophenylamino)-1-[2-(4-carbamoyl-2-nitrophenylamino)ethyl]propoxy ⁇ acetate (3.10 g, 5.82 mmol) in DMF (531 mL) was treated with 35% aq ammonia (9.7 mL) and then a solution of sodium dithionite (10.14 g, 58.2 mmol) in water (317 mL) was added dropwise at 0 °C. The reaction mixture was stirred at rt until consumption of the starting material was observed by LCMS. The resulting white precipitate was removed by filtration and discarded.
• Step 5 To a stirred solution of ethyl ⁇ 3-(2-amino-4-carbamoylphenylamino)-1-[2-(2-amino-4-carbamoylphenylamino)ethyl]propoxy ⁇ acetate (2.75 g, 5.82 mmol) in DMF (29 mL) was added 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (Intermediate 3; 2.5 g, 12.8 mmol) at 0 °C. The mixture was stirred for 30 min at 0 °C and formation of the thiourea intermediate was confirmed by LCMS.
• Building Block G-3L may be prepared from Building Block G-3 in a similar manner as described below for the synthesis of Building Block G-4L from Building Block G-4.
• amide coupling of Building Block G-3 with tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate followed by reaction with excess TFA affords Building Block G-3L.
• Step 1 To a stirred solution of Building Block G-4 (300 mg, 0.377 mmol) and tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate (121 mg, 0.377 mmol) in DMSO were added DIPEA (0.33 mL, 1.89 mmol) and HATU (144 mg, 0.377 mmol). The reaction mixture was stirred for 1 h and then purified by preparative HPLC (column: Waters Xbridge C18 250 mm x 19 mm, 5 ⁇ m; method: increasing gradient of 30% to 70% MeCN in water containing 10 mM ammonium bicarbonate at pH 9).
• Step 2 To a stirred solution of the product of Step 1 (266 mg, 0.242 mmol) in DCM (13 mL) was added TFA (2.5 mL) dropwise. The reaction mixture was stirred overnight at rt. The reaction mixture was concentrated under reduced pressure and the residue was freeze-dried, affording 15- ⁇ [(3- ⁇ 5-carbamoyl-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazol-1-yl ⁇ -1-(2- ⁇ 5-carbamoyl-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazol-1-yl ⁇ ethyl)propoxy)methyl]carbonylamino ⁇ -4,7,10,13-tetraoxapentadecanoic acid (Building
• Step 1 To a solution of Building Block G-4L (283 mg, 0.272 mmol) and 1-Boc-piperazine (51 mg, 0.274 mmol) in anhydrous DMSO (2.2 mL) was added DIPEA (0.24 mL, 1.38 mmol) followed by HATU (116 mg, 0.305 mmol). The reaction was stirred for 16 h at rt and then diluted and purified by preparative HPLC (column: SunFire C18, 250 mm x 19 mm, 5 ⁇ m; method: 30-70% MeCN in water containing 0.1% formic acid at pH 2).
• Step 2 The product obtained from Step 1 (254 mg, 0.210 mmol) was treated with DCM (2.3 mL) and TFA (1.2 mL). The resulting solution was stirred for 30 min and then concentrated under reduced pressure, affording the TFA salt of Intermediate 4 (266 mg, assumed 100 %) as a clear colourless gum.
• Step 1 To a stirred solution of N-[15-oxo-15-(1-piperazinyl)-3,6,9,12-tetraoxapentadecyl](3- ⁇ 5-carbamoyl-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazol-1-yl ⁇ -1-(2- ⁇ 5-carbamoyl-2-[(1-ethyl-3-methyl-5-pyrazolyl)carbonylimino]-3-methyl-2,3-dihydro-1H-1,3-benzimidazol-1-yl ⁇ ethyl)propoxy)acetamide TFA salt (Intermediate 4; 114 mg, 0.093 mmol) and 3-(3-(tert-butoxy)-3-oxopropoxy)propanoic acid ( CAS-no.
• Step 2 The product obtained from Step 1 (74 mg, 0.056 mmol) was treated with DCM (1.0 mL) and TFA (0.5 mL). The resulting solution was stirred for 1 h and then concentrated under reduced pressure, affording Intermediate 6 (71 mg, assumed 100 %) as a clear colourless oil.
• Step 1 To a stirred solution of Building Block G-4L (60 mg, 0.058 mmol) and tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecan-17-oate ( CAS-no. 1621616-14-5 ; 19 mg, 0.057 mmol) in dry DMSO (0.58 mL) was added DIPEA (0.060 mL, 0.344 mmol). After 10 min, HATU (24 mg, 0.063 mmol) was added. The reaction mixture was stirred at rt for 1 h.
• reaction mixture was purified by preparative HPLC (column: SunFire C18, 250 mm x 19 mm, 5 ⁇ m; method: 30-70% MeCN in water containing 0.1% formic acid at pH 2). Fractions containing the desired product were combined and concentrated under reduced pressure, affording the expected tert-butyl-protected intermediate (57 mg, 0.042 mmol, 74 %) as a clear colourless gum.
• Step 2 The product obtained from Step 1 (57 mg, 0.042 mmol) was dissolved in DCM (2.2 mL), and TFA (0.44 mL) was added. The resulting solution was stirred at rt overnight and then concentrated under reduced pressure, affording Intermediate 7 (55 mg, 100 %) as a colourless gum.
• reaction mixture was purified directly by preparative HPLC (column: Waters Xbridge C18 250 mm x 19 mm, 5 ⁇ m; method: 20-60 % MeCN in water containing 10 mM ammonium bicarbonate at pH 9). Fractions containing the desired product were concentrated giving a yellow film.
• Step 1 A solution of crude Building Block G-4 lithium salt (32 mg, assumed 0.034 mmol with purity 85 %) and tert-butyl N-[(1S)-1- ⁇ [(1S)-2-[(2S,4S)-4-(2- ⁇ 2-[2-(2-aminoethoxy)ethoxy]ethoxy ⁇ acetamido)-2- ⁇ [(1R)-1,2,3,4-tetrahydronaphthalen-1-yl]carbamoyl ⁇ pyrrolidin-1-yl]-1-cyclohexyl-2-oxoethyl]carbamoyl ⁇ ethyl]-N-methylcarbamate (25 mg, 0.0323 mmol) in DMSO was treated with DIPEA (0.028 mL, 0.1608 mmol) and stirred for 30 min.
• DIPEA 0.028 mL, 0.1608 mmol
• Step 2 To a stirred solution of the Boc-protected product of Step 1 (25 mg, 0.0161 mmol) in DCM (0.85 mL) was added TFA (0.22 mL). The reaction mixture was stirred at rt for 45 min and then concentrated under reduced pressure, redissolved in MeOH and concentrated again. The resulting residue was dissolved in MeOH and loaded to an SCX cartridge (sorbent bed weight 0.5 g). The cartridge was flushed with MeOH (3 CV) and then the product was eluted with 2M NH 3 in MeOH. The resulting eluate was concentrated under reduced pressure.
• the compound may be prepared similarly to Example 7 from Building Block G-3L and the corresponding commercially available amine ( CAS-no. 2446474-10-6 ).
• the compound is prepared similarly to Example 1 from Building Block G-3L and the corresponding commercially available amine.
• the compound is prepared similarly to Example 1 from Building Block G-3L and the corresponding commercially available amine.
• the compounds of the present invention are inhibitors of STING. As stated above, the compounds of the present invention are not STING agonists. Biological activities of the compounds of the present invention can be determined using any suitable in vitro assay as well as tissue and in vivo models.
• the STING-binding portion of the compounds of the invention are not STING agonists and may function as STING antagonists.
• CTD C-terminal domain
• recombinant human STING protein 155-379
• STING binding fluorescent probe were employed.
• the rotation of the fluorescent probe is slowed due to the larger molecular size of the protein-bound complex.
• emitted light retains its plane of polarization, leading to an increase in fluorescence polarization.
• Molecules competing for the probe binding site displace the probe, which leads to rapid randomization of the polarization plane of the emitted light and a subsequent decrease in measured fluorescence polarization signal. Change in polarization was measured, allowing a comparative evaluation of the binding affinities of different compounds to the target protein.
• the assay was performed in 96-well plates (ThermoFisher, cat.# 237108) containing 100 ⁇ l : 10 ⁇ L test compound in 10% DMSO and buffer A (150 mM NaCl, 50 mM HEPES pH 7.5, 1 mM DTT) + 90 ⁇ L prepared master mix solution of: 4 nM fluorescein-labelled STING ligand (Probe 1; synthesis described above) in buffer A + 0.055% Tween-20 (v/v) and 35 nM STING in buffer A. Post mixing, plates were agitated at 600rpm and incubated for 20 min at room temperature.
• Example number STING binding FP assay I IC50 (nM) 1 ⁇ 30 5 ⁇ 30 28 ⁇ 30 29 ⁇ 30 Building Block STING binding FP assay I, IC50 (nM) G-2 ⁇ 30 G-4 ⁇ 30
• STING binding FP assay II The procedure for STING binding FP assay II is the same as described for STING binding FP assay I, except for a lower STING concentration. In STING binding FP assay II, a concentration of 7 nM STING in buffer A was used.
• Example number STING binding FP assay II IC50 (nM) 5 2.4 7 3.0 8 2.5 9 8.4 12 2.1 14 5.7 15 1.7 16 4.5 17 7.9 29 1.8
• STING binding FP assay III The procedure for STING binding FP assay III is the same as described for STING binding FP assay I, except for a lower STING concentration. In STING binding FP assay III, a concentration of 10 nM STING in buffer A was used.
• Example number STING binding FP assay II IC50 (nM) 4 2.2 6 2.6 10 4.5 11 5.3 13 4.1 18 3.5 19 7.1 20 2.7 21 5.1 23 2.9 24 8.6 25 3.1 26 4.4 27 4.5 34 3.7 35 10.3 36 10.0 37 3.7 38 2.6
• CCD C-terminal domain
• the assay was run in CisBio low-volume white 96-well plates (catalog# 66PL96005) containing 5 ⁇ L compounds in Diluent Buffer (supplied with the CisBio kit).
• a solution of 1xSTING, 1x Anti-HIS-Tb and 1X D2-probe in TB detection buffer (kit supplied) was added to the plate. Plates were centrifuged for 2 min at 500 rpm, incubated for 60 min at room temperature in the dark, and then fluorescence emission at 665 nm following 330 nm xenon flash excitation on an BioTek synergy H1 multimode reader (Agilent) was measured.
• the plC50 values were determined using a standard four parameter curve fit in GraphPad Prism.
• HFF-1 cells (ATCC, SCRC-1041) cells were seeded at a density of 60.000 cells/well in in 24-well Nunclon Delta surface treated culture plate in DMEM growth medium (supplemented with 10% FBS, 1% Pen-strep). Cells were incubated over-night at 37°C 5% CO 2 to allow adhesion. Next day growth medium was renewed and a 5x concentration of test compounds (2.5% DMSO) diluted in growth medium were added to the appropriate well. The final DMSO concentration were 0.5%. Cell culture plates were returned to the incubator for additional 24h.
• cell culture plates were brought to ice, supernatant was removed by aspiration and cells washed 2x in ice-cold DPBS.
• Cells were lysed in 65 ⁇ L of RIPA buffer supplemented with cOmplete ULTRA Tablets protease inhibitor cocktail, PIERCE Phosphatase inhibitor mini tablet, Benzonase and 10mM NaF for 20 min on ice. Lysates were collected, cleared by centrifugation (13000xg for 10 min at 4°C). Samples were either analysed directly or stored at -80°C until analysis.
• total protein concentration in each lysate was determined using PIERCE Gold BCA Assay kit according to manufactures protocol. 6 ⁇ g of total protein was mixed with 2x Laemmli buffer containing DTT, boiled for 5 min at 95°C, cooled on ice and loaded into each well of a 4-20% Criterion TGX gel. Proteins were separated by electrophoresis, by running for 1h at 150V. After separation proteins were blotted onto a PVDF membrane by semi-dry transfer using Trans-Blot turbo system at pre-defined BioRad MIDI protocol; 25V, 1A (fixed) for 30 min.
• Membranes were blocked for 1h in 5% skim milk in TBS-T20 on a rocker at room temperature followed by washing and exposure to primary antibodies against human STING 1:1000 (Cell Signalling, #13647) or loading controls Vinculin 1:1000 (Cell Signalling, #13901) or beta-Actin 1:10000 (Sigma, A5441) all diluted in 5% BSA in TBS-T20.
• Membranes were probed over night at 4°C on a rocker.
• Membranes were washed 3x in TBS-T20 followed by exposure to secondary HRP-conjugated anti-rabbit antibody or HRP-conjugated anti-mouse antibody diluted 1:10000 in 5% skim milk in TBS-T20 as appropriate.
• Membranes were incubated for 1h at room temperature on a rocker, then washed 3x in TBS-T20. Signals were revealed using clarity ECL mixed 1:1 for 1 min and imaged using ImageQuant800.
• the intensity of individual bands was measured using imaged and expression levels were calculated as a ratio of STING intensities vs. Reference protein intensities and normalized to DMSO control. The normalized ratios were plotted on a XY curve in GraphPad Prism where DC 50 was estimated using a 4-parameter non-linear regression fitting curve.
• Example number STING degradation in HFF-1 cells DC50 (nM) Reference protein used for DC50 determination 3 ++ beta-actin 6 ++ beta-actin 10 +++ vinculin 11 +++ beta-actin 16 +++ beta-actin 23 ++ beta-actin 24 ++ vinculin 25 +++ beta-actin 26 +++ vinculin
• H596 cells ATCC, HTB-178 cells were seeded at a density of 100.000 cells/well in in 24-well Nunclon Delta surface treated culture plate in RPMI-1640 growth medium (supplemented with 10% FBS, 1% Pen-strep). Cells were incubated over-night at 37°C 5% CO 2 to allow adhesion. Next day growth medium was renewed and a 5x concentration of test compounds (2.5% DMSO) diluted in growth medium were added to the appropriate well. Final DMSO concentration was 0.5%. Cell culture plates were returned to the incubator for additional 24h. Final test compound concentrations were 0.22 ⁇ M, 0.67 ⁇ M, 2 ⁇ M and 6 ⁇ M.
• Example compounds 14, 16 and 24 demonstrate induction of STING degradation using either compound.
• Example compounds 16 and 24 showed a more potent effect of induction of STING degradation than Example compound 14, with Example compound 14 inducing visible degradation in H1650 and H596 at the higher concentrations tested but did not induce visible STING degradation in HaCat cells.
• Example number STING degradation in H596 cells DC50 (nM) Reference protein used for DC50 determination 14 + beta-actin 19 +++ beta-actin 20 +++ beta-actin 27 +++ beta-actin
• Example compounds 14, 16 and 24 Similar protocols were carried out to assess STING degradation in H1650 cells ( Figure 1 , top panel) and HaCat cells ( Figure 1 , bottom panel) using Example compounds 14, 16 and 24, except that the seeding density was 150,000 cells/well and 250,000 cells/well for H1650 cells and HaCat cells, respectively. As shown in H596 cells, Example compounds 14, 16 and 24 induced STING degradation, with Example compounds 16 and 24 having a more potent effect than Example compound 14.
• Figures 2A-C demonstrate that each compound tested induced STING degradation in mouse cells in a dose-dependent manner.
• Example 46 Western Blot analysis for STING recovery following Example compound washout in HFF-1 cells
• HFF-1 cells were treated with 1 ⁇ M Compound E16 or E24 or left untreated (UT) for 24h prior washout of test compound, and then incubated for 0-48 hours in DMEM without test compound. At the different time points cell lysates were prepared and assessed by western blot using anti-STING (1:1000) and anti-VCL (1:1000) antibodies. Controls included cells left untreated.
• Figure 3 shows that following incubation with either Example compound 16 or Example compound 24, STING levels failed to recover to pre-treatment levels after 48 hours following removal of Example compound. Slow rebound of STING protein after washout suggests the intracellular half-life of the Example compounds will allow for a prolonged protein degradation effect and that this prolonged effect will be seen after in vivo exposure.
• Example 47 Western Blot analysis for STING degradation in STING-associated vasculopathy with onset in infancy (SAVI) patient cells
• STING degradation was assessed in primary fibroblast cells with constitutively active STING (comprising a N154S mutation), derived from SAVI patients, and compared to healthy donor primary fibroblast cells.
• Cells were seeded at 50,000 cells/well and cultured under standard conditions for 24 hours. Media was replaced with test compound and incubated for 20 hours prior to analysis.
• Figure 4 shows Example compounds 16 and 24 induced STING degradation in health donor cells and SAVI patient cells, demonstrating that these compounds can induce degradation of not only wild-type STING but constitutively active STING expressed in patients.
• Figure 5 shows the results of Western Blots analysis of human skin fibroblasts treated with 5 ⁇ M of A9 or A11 compound either before, simultaneously with, or after activation with the 0.3 ⁇ M diABZI.
• Cells were incubated with A9 or A11 for 1 or 2 hours before addition of diABZI c3, treated with test compound and diABZI c3 simultaneously, or treated with A9 or A11 after 15 minutes incubation with diABZI c3.
• As a control cells were also treated with diABZI c3 alone.
• Figure 5 shows that under all conditions tested, A9 and A11 inhibited activation of STING, as demonstrated by a reduced intensity of P-STING.
• the downstream effector of active STING, TBK1 also showed reduced levels of activation as demonstrated by reduced P-TBK1 intensity compared to cells only treated with diABZI c3.
• activation of the downstream effector LC3B is also reduced following treatment with Compounds A9 and A11 as demonstrated by reduced LC3B lipidation.
• HFF-1 cells were treated with 5 ⁇ M of compounds A11, A25, A26, or A30, or 0.5 ⁇ M STING antagonist control for 15 min followed prior to addition of 300 nM diABZI and incubation for 2 h or 5 h.
• the cells were lysed and analysed on WB.
• cells were left untreated (UT), treated with Lipofectamine (Lipo control) or with compounds A26 or A30 as single agent.
• the WB membrane was probed for p-STING, STING, p-IRF3, p-TBK1, and Vinculin.
• Example 49 Building Blocks act as STING antagonists and do not act as STING agonists: Human FB STING antagonist assay
• Example 50 - Example compounds do not act as STING agonists
• Human fibroblast were treated with a titration of E22, E9, E14 and E16 (7.2 ⁇ M-3.6nM) for 24h prior to cell lysis and subsequently total STING, pSTING and Vinculin were assessed by western blot using rabbit anti-STING (1:1000), anti-pSTING (1:1000), anti-Vinculin (1:1000) and 2nd anti-rabbit-HRP (1:10.000) or anti-mouse-HRP (1:7500). Proteins were visualized using ImageQuant800. Control cells included untreated cells and cells treated with 300nM diABZI C3.
• Example 51 Inhibition of STING-agonist-induced IFN- ⁇ release in HFF-1 cells (EC50) and mouse RAW264.7 cells
• test compounds were assessed inhibition of STING-agonist-induced IFN- ⁇ release in human foreskin fibroblasts HFF-1 cells (ATCC, SCRC-1041).
• Cells were seeded at a density of 10.000 cells/well in in 96-well Nunclon Delta surface treated culture plate in DMEM growth medium (supplemented with 10% FBS, 1% Pen-strep). Cells were incubated over-night at 37°C 5% CO 2 to allow adhesion. Next morning growth medium was renewed and a 7x concentration 3-step dilution series of test compounds (2,1% DMSO) diluted in growth medium were added to the appropriate well. Final DMSO concentration was 0.3%.
• GSK STING agonist CAS number: 2138299-34-8 ; diABZI c3; diABZI
• final concentration was 0.3 ⁇ M.
• Cells culture plates were further incubated, and supernatants were harvested after 6h and immediately stored at -20°C until determination of IFN- ⁇ release.
• Raw data values were quantified based on the standard curve using blank subtracted ⁇ 450nm-570nm values generated using the Gen5 software connected to the plate reader. Concentrations were plotted into GraphPad Prism to calculate EC 50 values using a 4-parameter non-linear regression fitting curve, bottom constrains set for >0.
• Example number Inhibition of STING-agonist-induced IFN- ⁇ release in HFF-1 cells EC50 (nM) 14 + 16 +++ 24 ++* * incomplete inhibition
• Figure 8 also demonstrates that Example compounds disclosed herein inhibit STING-agonist induced IFN- ⁇ release in mouse RAW264.7 cells.
• RAW264.7 cells treated with 1.8 ⁇ M diABZI c3 increased IFN- ⁇ secretion, resulting in a media concentration of around 1000 pg/mL.
• cells treated with 1.8 ⁇ M diABZI c3 and 0.05 ⁇ M Example compounds 14, 16 or 24 reduced this expression to approximately 500 pg/mL. This inhibitory effect was even more pronounced in cells treated with either 0.5 ⁇ M or 5 ⁇ m of either one of Example compounds 14, 16 or 24.
• HFF-1 cells were also assessed for inhibition of STING-agonist-induced IFN- ⁇ and CXCL10 release in cells treated with Compounds A9 and A11.
• cells treated with only diABZI c3 resulted in ⁇ 200 U/mL IFN media concentration.
• pre-treating cells for 1 to 2 hours prior to diABZI c3, simultaneous treatment with A9 and diABZI c3, or pre-treating cells for 15 minutes with diABZI c3 followed by A9 treatment led to a decrease in the IFN media concentration to ⁇ 100 U/mL.
• Example number Inhibition of STING-agonist-induced IFN- ⁇ release in H596 cells EC50 (nM) 10 ++ 16 + 19 +++ 20 +++ 24 +++
• Example 53 Pharmacokinetic profiling of Example compounds in mouse
• Example compound 16 ( Figure 10A ) or 8 mg/kg Example compound 24 ( Figure 10B ) by subcutaneous injection. Plasma concentration of test compound was measured over the course of 8 hours.
• Example compound 16 Subcutaneous injection with either Example compound 16 or Example compound 24 leads to prolonged plasma exposure in mice. 8 hours after injection, the plasma concentration of Example compound 16 is approximately 100 nM, while the plasma concentration of Example compound 24 is approximately 1000 nM. These data are also consistent with plasma concentrations following intravenous injection which show a lower clearance rate of Example compound 24 vs Example compound 16 after 8 hours.
• IFN- ⁇ and CXCL10 (IP-10) levels were analysed 6 hours after treatment with diABZi.
• One outlier in mice receiving pre-treatment with Example compound 16 was present in treatment arm iii) and is shown in Figures 11B and 11C as an open circle.
• FIG 11B shows that in mice pre-treated with either Example compound 16 or Example compound 24 IFN- ⁇ in dose arm i), IFN- ⁇ expression appears to be completely abolished. Similar results were seen in mice treated with SP-0150 in dose arms ii) and iii), with IFN- ⁇ expression IFN- ⁇ expression appearing to be completely abolished. Similar, but slightly less pronounced results, are observed in mice treated with Example compound 16 in dose arms i) and ii) and show a significant inhibition of IFN- ⁇ .
• Figure 11C shows that in mice pre-treated with Example compound 16 in each dose are i)-iii) show significantly reduced CXCL10 expression. Similarly, mice pre-treated with Example compound 24 in each dose arm also show reduced CXCL10 expression, with dose arm ii) reaching statistical significance.
• the S1 portion of the above compounds (e.g. Building blocks G-1, G-2 and G4, and Compounds A9, A11, A25, A26 and A30) have been demonstrated to bind to STING and, in the case of G-1, G-2, Compounds A9, A11, A25, A26 and A30, do not act as STING agonists.
• Each S1 region of the Example compounds of the invention share the core of at least one of G-1, G-2, A9, A11, A25, A26 and A30 chemical structure. Viewing the data as a whole, it is clear that the compounds of the invention have a prolonged STING protein degradation effect, which is consistent with the PK profiles discussed above.
• Example compounds also do not act as STING agonists. This is supported by the data in Figure 7 which demonstrates that none of Compounds E9, E14, E16 or E22 induce STING phosphorylation.

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